Bio-oxidation of steroids



i atentecl Nov. 3, 1953 BIO-OXIDATION F STEROIDS William J. Haines and Marvin H; Kuizenga, Kalamazoo, Mich., assignors to. The Upjohn; Company, Kalamazoo, Mich a corporation, of

Michigan No Drawing; Application August 19, 1950, Serial No. 180,508

22 Claims. 1

The present invention relates to amethod for the biological oxidation of steroids wherein the bio-oxidation is accomplished by the employment of glandular tissue, or enzymes obtainable therefrom. More particularly, the invention relates to the biological introduction of oxygen into the eleven position of a steroid molecule by means of mammalian adrenal glands or enzymes thereof: or present therein. The present application is a continuation-in-part of our prior-filed copending application Serial. No. 690,074, filed August 12, 1946.

It is known that the adrenal cortex contains certain steroids which contain an oxygen atom in the eleven position. While other steroids, not containing the eleven oxygen atom, are also found in the adrenal cortex, these eleven desoxy steroids are of small import, since they do not possess the unusually high physiological activity which is characteristic of the eleven oxygenated compounds. These eleven desoxy steroids, moreover, exhibit practically no effect when tested for physiological emcacy in the well-known Ingle work test [Endocrinology 26, 472 (1940)], while studies of the relative efficacy of various steroids in the Ingle work test uniformly support the conclusion that the eleven oxygenated adrenal steroids are of great potency in this test. LIngle, Endocrinology 26, 472 (1940); Proc. Soc. Exper. Biol. and Med. 44. 450 (-1940); Am. J. Physiol. 133, 676 (1941); Endocrinology 34, 19-1 (1944); Ingle and Kuizenga, Endocrinology 36, 218 (1945-); Ingle, Pabst, and Kuizenga, Endocrinology 36, 426 (1945).] For example, large doses of ll-desoxycorticosterone, of from four to twenty milligrams daily, have been found to exert only a small, almost negligible, effect upon work performance in the test, while only fractions of a milligram of the eleven oxygenated adrenal cortex steroids have at least the same potency. In a test of ll-desoxycorticosterone, reported in Am. J. Physiol. 133, 6'76- (1941), this compound was found to exhibit little or no activity in the work test in doses up-to four milligrams daily. Since this is true, and since results of isolation studies show a close correlation between (a) the number of work units. in a given test fraction and (b) the recovery of eleven oxygenated adrenal steroids therefrom lKuizenga, Wick, Ingle, Nelson and Cartland, J. Biol. Chem. 147, 56.1 (1943) it follows that the activity or potency of adrenal cortical tissue extracts, as measured by the Ingle work test, is directly relatable to the quantity of eleven oxygenated cortical steroids. contained in the extracts. No data being available to the, con,-

trary, it was the observation of this. factor, the direct correlation between potency, as shown by the Ingle work test, and the quantity of eleven oxygenated adrenal steroids. in the test extracts, which led us to the discovery of the present invention, viz., that eleven desoxy cortical steroids can :be converted into eleven oxygenated adrenal steroids by the action of mammalian adrenal glands, or enzymes present therein.

The use of the eleven oxygenated adrenal cortex steroids in. therapy has now increased to such an extent that the total supply of mammalian adrenal glands necessary for their production has become a limiting factor in the amount of eleven oxygenated adrenal cortex steroids available for use. As the. animal organism apparently doesnot store adrenal cortex hormones, it has been indicated: that only a relatively small amount. of these hormones exists at any one time in the-cortical tissue of mammalian adrenal glands. Only the amount of the adrenal cortex hormone existing in the gland at the time of death, lessv any loss in processing, has been previously available from well preserved glands. Further, as the adrenal gland represents. but a small part of the total body-weight of the animal, and is accordingly a byproduct, rather than a prime factor, in the determination of the animal population or the number of animals to be slaughtered commercially, the demand for adrenal glands cannot stimulate the supply of animals from which they are obtained. The users of adrenal glands. have therefore no method at their disposal to increase the supply of adrenal glands as such. At the same time, the unusual utility of those adrenal cortical steroids having an oxygen atom in the eleven position, such as corticosterone, 1l-dehydrocorticosterone, li-dehydro-17-hydroxycorticosterone (Compound E, cortisone), and ,l7-hydroxycorticosterone (Cornpound F) has placed these compounds in everincreasing demand. All of these compounds show physiological activity when tested. by the Ingle work test supra, or by liver glycogen deposition assay [Pabst etaL, Endocrinology 41, (1947) 1, while related adrenal steroids without the eleven oxygenation, such as, ll-desoxycorticosterone and l7-hydroxydesoxycorticosterone (Substance S), and the like, show substantially no activity by these tests. It is obvious that the eleven oxygenated adrenal cortical steroids, because of their unique physiological activity, are of great value, and these, compounds, as well as methods for the production thereof, have been the subject of extensive research,-at present withno avail. While processes for introducing oxygen into the eleven position of a steroid molecule by chemical means have been developed, such procedures require many steps and are consequently time-consuming and expensive. The importance of a new and efficient method for the production of eleven oxygenated steroids, which can be adapted to large scale production, as is true of the method of the present invention, can not be overemphasized. This importance is undoubtedly most obvious to members of the general public who presently suffer from physiological abnormalities which are known to be remedied or at least beneficially affected only by certain of the eleven oxygenated adrenal cortex steroids, which compounds are presently generally unavailable due to the lack of satisfactory methods for their production and a consequent inadequate supply, which factors combine to place these drugs beyond the reach of the average individual due to the prohibitive expense involved.

It is an object of the present invention to provide a method for the introduction of oxygen into the eleven position of a steriod nucleus which involves bio-oxidation of the eleven desoxy steroid by means of mammalian adrenal gland tissue or enzymes present therein. It is a further object of the present invention to provide a method for the introduction of oxygen into the eleven position of an eleven desoxy adrenal cortex steroid, whether naturally present in the adrenal cortex tissue, or whether added from an exterior source, by the action of mammalian adrenal gland tissue, or enzymes present therein. An additional object of the invention is the provision of such process-which involves incubation of the mammalian adrenal gland tissue together with the eleven desoxy steroid, whether naturally occurring in the gland tissue or added thereto. Another objective is to provide such process involving the use of aeration, and a still further objective to provide such method wherein comminuted gland is employed in water suspension or in a nutrient medium. Still another object of the invention is to provide such a process wherein cofactors, aiding oxidation in the eleven position of the steroid nucleus,

are employed together with the glandular tissue.

Other objects will become apparent hereinafter. The term eleven desoxy steroid, as used herein, refers to a steroid which contains no oxygen at the eleven position. v

The method of the present invention consists in incubating together mammalian adrenal gland tissue and an eleven desoxy steroid, i. e., one containing no oxygen in the eleven position, and recovering the resulting eleven oxygenated steroid from the reaction mixture. The eleven desoxy steroid can be that normally occurring in the mammalian adrenal cortex, or it can be procured from external sources and added to the incubation reaction mixture. The mammalian adrenal gland can be that of cattle, sheep, or hog, with hog adrenal glands being preferred.

In carrying out the process of the present invention, the whole, fresh, frozen adrenal glands of slaughterhouse animals are first comminuted by any suitable means. This is usually accomplished with a meat grinder of suitable size, although any other means of comminution may be employed. The glands are preferably frozen at a temperature below about minus thirty degrees centigrade substantially immediately after isolation from the animal body. This may be accomplished in any number of ways, as, for example, by storing the gland, immediately upon removal from the animal, in a, container cooled with liquid nitrogen or solid carbon dioxide, although other methods of cooling the gland to below about zero degrees centigrade are satisfactory. It is highly desirable that the gland be maintained at such lower temperature until used in the process of the invention, since the glands can thus be stored until a sumcient quantity for large scale use has been accumulated and can also be transported considerable distances in this condition. Moreover, the activity of the gland in the process of the invention has been found to be enhanced where the gland is frozen immediately upon removal from the freshly slaughtered animal.

The ground adrenal gland and the steroid material to be oxygenated in the eleven position are intimately admixed, as by vigorous agitation or homogenization in a Waring-type blender or other grinding device. The mixture may thereafter be admixed with a suitable medium, or, alternatively, the ground adrenal gland and the steroid material may be admixed with the medium prior to the blending operation. The mixture is thereafter maintained preferably at or about 37.5 degrees centigrade, i. e., body temperature, but temperatures between about twenty and forty degrees centigrade are also operative. The medium employed for the oxygenation reaction, which has been previously referred to, is an aqueous nutrient medium, which may consist of body fluids, for example, blood or plasma, or a slightly alkaline physiological saline solution such as Krebs-Ringer solution made alkaline to a pH of about 7.4, as with sodium bicarbonate, or a Ringer-phosphate bufier solution, or other physiological saline solutions, or an aqueous solution of water containing bacteriostatic quantities of alcohol, e. g., up to about twenty percent alcohol, or even water containing traces of minerals and other elements such as are commonly present in tap water, which of itself provides a satisfactory nutrient medium for carrying out the process of the present invention. The pH of the medium employed is preferably slightly alkaline, e. g., about 7.4, but a medium having a pH anywhere in the range between about 6.5 and 8.0 is also satisfactory. Cofactors, such as those containing an adenine or nicotinamide nucleus, may also be included in the medium, and such cofactors are preferably included since the yields of oxygenated cortical steroids, as shown by the glycogen deposition test of Pabst, et al., Endocrinology 41, 55 (1937), appears to be increased when such cofactors are present. Representative cofactors which can be used include nicotina-mide, cytochrome C, sodium adenosine triphosphate, Coenzynie I (diphosphopyridine nucleotide), FAD (fiavin-adenine dinucelotide), adrenocorticotropichormone (ACTH), and the like, with nicotinamide being preferred for highest yields of eleven oxygenated steroid.

The incubation is carried out under aerobic conditions, i. e., in the presence of oxygen, and for this reason it is necessary that the reaction mixture be, at a minimum, stirred occasionally to provide oxygen from the air. While such procedure as occasional stirring is operative as a means of providing aerobic conditions, it is preferred to pass an oxygen-containing gas into the reaction mixture through a suitable means, such as fritted glass discs, carborundum balls, sparger tubes, Berkfeld filter candles, and the like. The y r t o y en-containin gas/can be passed into the reaction mixture in any quantities sufiicient to maintain aerobic conditions within the incubation medium, and the rate of aeration may therefore be varied considerably, depending particularly upon the amount of glands employed or the size of the oxygenation run. However, it has been found that between about 0.1 and about five liters of oxygen is satisfactory, between about 0.5 and about five liters of oxygen per liter of solution being preferred. The employment of a diluent, such as carbon dioxide, nitrogen, helium, or the like, together with the aerating oxygen, apparently has no adverse effect upon operativeness of theprocess. The time of incubation will vary considerably, depending upon the rate of aeration and source of the steroid to be oxygenated, as well as the ratio of ground gland to the steroid being oxidized. While periods of from two to five hours are in most instances satisfactory for the conversion of eleven desoxycortical steroids to eleven oxygenated cortical steroids when the steroid is added to the glands from external sources, with longer periods being operative but of no advantage, it should be apparent that, when the steroid to be oxygenated is present only in the adrenal cortex tissue, none being added to the comminuted gland from external sources, the time required for oxygenation may be considerably longer, perhaps due to the integral relation of these steroids to the tissue fibers or other constituents of the gland tissue. Therefore, in instances when the steroid to be oxygenated is not added from external sources but is merely that amount present in the adrenal cortex of the glands which are used for the incubation procedure, the time of reaction may advantageously be conducted for periods as long as forty, and even as long as 144 hours. It is to be noted, however, that exceedingly vigorous aeration over an extended period of time appears to have a somewhat detrimental effect upon the efficiency of the process, although complete inoperativeness of the process is probably not attained until considerably after the l44-hour period mentioned. Other variable factors will also influence the conversion and yield of eleven oxygenated steroid, such as the season of the year in which the gland was collected, average state of health of the animal from which the glands were collected, the particular cofactors present in the nutritive medium, theparticular nutritive medium employed, and the like.

After a suitable period of incubation, the oxygenated cortical steroids produced by the process of the invention can be isolated and identified by known procedures for the separation of such materials from adrenal glands. Any one of the previously known methods for the isolation of adrenal cortical hormones may .be employed, such as, for example, that described in the J our-- nal of Biological Chemistry 147, 561 (1943) or in U. S. Patent 2,528,880, issued on application Serial 766,884, filed August 6, 1947. For many purposes mixtures of these adrenal cortical steroids, as produced by theprocess of the invention, are entirely satisfactory so that it is not essential to separate the individual components of the mixture of eleven oxygenated steroids. However, if it is desired to separate the chemical individuals, procedures are known in the art whereby such can be accomplished.

In the event that an aqueous. alcohol solution is employed as a nutrient medium, together with aeration, it is often advantageous to addzadditional alcohol at a later stage in the process to:

replace that lost by evaporation. Moreover, in the event that a somewhat lengthy reaction period is desired to be employed, e. g., a reaction period from about forty to 144 hours, the addition of an agent to prevent putrefaction of the glands may be advantageously employed if desired. Bacteriostatic agents, such as alcohols, toluene, benzene, benzoates, quaternary ammonium salts, chlorobutanol', and the like, as well as adjustment of the pH to within the desired range, and other known procedure for the prevention of putrefaction, may be employed to accomplish the desired result. A convenient method of preventing putrefaction is the employment of an aqueous alcoholic medium, containing up to about twenty percent alcohol, in which case. the alcohol appears to serve both asv a part of the nutrient medium and as an agent for prevention of putrefaction. As mentioned previously, if aerating conditions are used during the incubation, additional alcohol may be advantageously added to replace that lost by evaporation.

Steroid starting materials which are suitable for the method of the present invention include the eleven desoxy steroids, i. e., those steroids having no oxygen in the eleven position, which are oxidizable to an eleven oxygenated steroid. This includes numerous members of the cyclopentanopolyhydrophenanthrene series, especially those compounds containing an eleven methylene group. Representative steroid compounds which are suitable starting materials in the method of the present invention include. ll-desoxycorticosterone, progesterone, 3-hydroxypregnane-20-one, 3-hydroxydelta 5'-pregnen-20-one, l7-hydroxyprogesterone, ll-desoxy-l'l-hydroxycorticosterone, and various acylated and alkylated derivatives thereof. The ratio of steroid to gland, or vice versa, does not appear critical, since weight ratios of gland to steroid of as little as fifty to one and as high as 2000- to one have proved satisfactory for the conversion of eleven desoxy cortical steroids to eleven oxygenated steroids. However, ratios even lower or higher are also operative, ratios as low as ten parts of gland by weight to one part of steroid being operative, although perhaps notcommercia-lly feasible since the percent conversion and yield of eleven oxygenated steroid appears to vary directly with the ratio of gland to steroid employed. Of particular interest as starting materials, whether added to the comminuted adrenal glands or whether present in the adrenal cortex tissue, may be mentioned especially the compounds ll-desoxycorticosterone, ll-desoxy- 17-hydroxycorticosterone (S), and l7-hydroxyprogesterone, which compounds are known to exist in adrenal cortex tissue. These compounds are convertible to the more highly active eleven oxygenated cortical steroids by the method of the present invention, e. g., ll-desoxycorticosterone is converted to corticosterone and 'll-desoxy-l'l hydroxycorticosterone (compound S) is converted to 17-hydroxycorticosterone (compound F).

As mentioned .in the. foregoing, since the eleven desoxysteroids. show no activity of any significanoe when measured by either the. work test of Ingle or the glycogen deposition assay test of Pabst, et al., the increase in activity of the. adrenal cortical extracts as shown by the Ingle work test or the Pabstglycogen deposition assay test has accordingly been used to show the converacospaaw sion of eleven desoxy cortical steroids to corresponding eleven oxygenated steroids by the process of the present invention, the eleven oxygenated steroids being known to exhibit considerable potency when measured by these tests.

The following examples are to be understood as illustrative only, and are in no way to be construed as limiting the invention.

Example 1 The processing of 3175 kilograms of adrenal glands according to methods known in the art [J. Biol. Chem. 147, 561 (1943)], is known to give an average yield of fifty units per kilogram of gland as determined by the work performance test of Ingle, mentioned previously. This figure of fifty units is based on amount of activity found in practice over an experience of many years.

Control One hundred six kilograms of frozen hog adrenal glands were divided into lots of 100 kilograms and six kilograms, respectively. The 100- kilogram lot was ground and mixed with acetone and the adrenal cortex hormones isolated by the procedure referred to above. Forty four work units per kilogram of gland were obtained.

The six-kilogram lot was ground, suspended in six liters of water and divided into two equal portions which were treated as follows:

Example 1a Six hundred milliliters of 95 percent ethyl alcohol, a bacteriostatic agent, Was added to three kilograms of ground, frozen hog adrenal glands suspended in three liters of water. The slurry was then placed in a glass-lined can and allowed to incubate for a period of 120 hours, with occasional stirring, at a temperature which varied between 25 and 30 degrees centigrade. At the end of this time about 3.5 volumes of acetone was added and the cortex hormones isolated by the method to which reference has previously been made. On the basis of the work test, already mentioned, the activity of the adrenal cortex hormones isolated equalled 430 units per kilogram of gland, indicating that a considerable portion of ll-desoxy adrenal cortical steroids had been converted into ll-oxygenated adrenal cortical steroid hormones.

Example 1?) Six hundred milliliters of 95 percent ethyl alcohol, a bacteriostatic agent, was added to three kilograms of ground, frozen hog adrenal glands suspended in three liters of water. The slurry was then placed in a glass-lined can and allowed to incubate for a period of 120 hours, with occasional stirring, at a temperature which varied between 25 and 30 degrees Centigrade. During this time a continuous stream of air was introduced into the slurry through two Berkfeld candles. At the end of 65 and 96 hours, one liter of ten percent alcohol in water was added for bacteriostatic purposes. At the end of 120 hours, about 3.5 volumes of acetone was added and the hormones isolated. There was obtained 1341 units of material per kilogram of gland, as shown by the Ingle work test, indicating that a considerable quantity of ll-desoxy adrenal cortical steroids had been converted to ll-oxygenated adrenal cortical steroids by the incubation procedure. r

8 r Example 2 Twelve kilograms of hog adrenal glands were ground while frozen and mixed with twelve liters of water. Two and four-tenths liters of ethyl alcohol were added for bacteriostatic purposes, and the suspension divided into four equal portions, each portion being placed in a glass bottle of five-gallon capacity. Each portion was then treated in the manner given below:

Example 2a The suspension of the gland was incubated for a period of 48 hours at a temperature which varied between 23 and 25 degrees centigrade. During the entire period of incubation a stream of air was bubbled through the suspension by means of a coiled rubber tube having holes therein and located at the bottom of the bottle. Air was passed through the suspension at the rate of 1.6 liters per minute per kilogram of gland (actual rate, five liters per minute). At the end of the incubation period, 3.5 volumes of acetone was added and the hormones isolated. As shown by the Ingle work test, 83 units per kilogram of gland was obtained, which is considerably more than the average of 50 units usually obtained, indicating considerable conversion of ll-desoxy adrenal cortical steroids to ll-oxygenated adrenal cortical steroids by the incubation procedure.

Example 2b The suspension of the gland was incubated for a period of 144 hours under conditions as given in Example 2a. Three and one-half volumes of acetone was added and the hormones isolated. As shown by the work test, there was obtained 62 units per kilogram of gland, considerably more than the usual fifty units, which shows some conversion of ll-desoxy adrenal cortical steroid to ll-oxygenated adrenal cortical steroid by the incubation step.

Example 2d The suspension of the gland was incubated for a period of 144 hours at a temperature which varied between 23 and 25 degrees centigrade. The suspension was allowed to incubate without forced aeration. Three and one-half volumes of acetone was added and the hormones isolated. As shown by the work test, there was obtained 73 units per kilogram of gland, considerably more than the usual fifty units, which shows some conversion of ll-desoxy adrenal cortical steroid to ll-oxygenated adrenal cortical steroid by the incubation procedure.

Example .3

A suspension was prepared of six kilograms of ground hog adrenal glands in six liters of water and 1.2 liters of alcohol, added for bacteriostatic purposes. The suspension was divided into two equal portions, placed in five-gallon glass bottles, and treated as follows:

9 Example 3a The suspension was incubated for ninety hours at a temperature of 23 to 25 degrees centigrade. During the entire incubation period, air was passed through the suspension, using carborundum balls, at a rate of five liters of air per minute per kilogram of gland (actual rate, fifteen liters per minute). At the end of the incubation period, 3.5 volumes of acetone was added and the hormones isolated. As shown by the Ingle work test, there was obtained 62 units per kilogram of gland, considerably more than the usual fifty units, showing conversion of some ll-desoxy adrenal cortical steroids to ll-oxygenated adrenal cortical steroids by the incubation procedure.

Example 317 The suspension was incubated for ninety hours at a temperature of 23 to 25 degrees centigrade. Three or four times daily the incubating suspension was stirred vigorously for a short time. At the end of the incubation period, 3.5 volumes of acetone was added and the hormones isolated. As shown by the Ingle work test, there was obtained 115 units per kilogram of gland, considerably more than the usual fifty units, showing conversion of ll-desoxy cortical steroid to 11- oxygenated adrenal cortical steroid by the incubation procedure.

Example 4 A suspension was prepared of twelve kilograms of ground hog adrenal glands in twelve liters of water and 2.4 liters of alcohol, added for bacteriostatic purposes. The suspension was equally divided into three portions and each portion was placed in a five-gallon glass bottle. The suspension was agitated throughout the incubation period. Further treatment of two of the portions was as follows:

Example 4a The incubation was conducted at 38 degrees centigrade for a period of 96 hours. Air was passed into the suspension through fritted glass discs at a rate of about 1.2 liters per kilogram of gland per minute (actual rate, five liters per minute). At the end of the incubation period, acetone was added and the hormones isolated. As shown by the Ingle work test, there was obtained 190 units per kilogram of gland.

Example 4b The incubation time and temperature was the same as in Example 4. Aeration, through fritted glass discs, was at a rate of 2.5 liters per minute per kilogram of gland (actual rate, ten liters per minute). At the end of the incubation period, acetone was added and the hormones isolated. As shown by the work test, there was obtained eighty units per kilogram of gland, considerably more than the usual fifty units, showing considerable conversion of ll-desox-y adrenal cortical steroids to ll-oxygenated adrenal cortical steroids by the incubation procedure.

Example 5 A suspension was prepared of twelve kilograms of ground hog adrenal glands in twelve liters of water and 2.4 liters of alcohol, added for bacteriostatic purposes. The suspension was divided into two equal parts, each part being placed in a five-gallon glass bottle equipped with an agitator. Agitation was maintained throughout the incubation period. Further treatment was as follows Example 5a The incubation was conducted at a temperature of 38 degrees centigrade for a period of 96- hours. Aeration, through iritted glass discs, was maintained at a rate of 0.8 liter per minute per kilogram of gland (actual rate, five liters per minute). One liter of ten per cent alcohol was added after 24 hours and again after 48 hours, to maintain bacteriostatic conditions. At the end of the incubation period, acetone was added and the hormones isolated. As shown by the work test, there was obtained 155 units per kilogram of gland, considerably more than the usual fifty units, showing considerable conversion of '11- desoxy adrenal cortical steroids to ll-oxygenated adrenal cortical steroids by the incubation procedure.

Example 5b The incubation was conducted at a temperature of 38 degrees centigrade for a period of 96 hours. Aeration, through fritted glass discs, was maintained at a rate of 0.4 liter per minute per kilogram of gland (actual rate, 2.5 liters per minute). As shown by the work test, there was obtained 158 units per kilogram of gland, considerably more than the usual fifty units, showing high conversion of 11-desoxy adrenal cortical steroid to ll-oxygenated adrenal cortical steroid by the incubation procedure.

Example 6 Two hundred kilograms of frozen hog adrenal glands were ground into a mixture of 200 liters of water and twenty liters of alcohol, added for bacteriostatic purposes. Incubation was conducted at temperatures between twenty and thirty degrees centigrade for a, period of hours. Aeration, using a stainless steel sparger, was maintained at a rate of 1.2 liters per minute per kilogram of gland (actual rate, 500 cubic feet per minute). At the end of the incubation period, acetone was added and the hormones isolated. As shown by the work test, there was obtained 165 units per kilogram of gland, considerably more than the usual fifty, which shows considerable conversion of ll-desoxy adrenal cortical steroid to ll-oxygenated adrenal cortical steroid by the incubation procedure.

Example 7a Hog adrenal glands were frozen immediately after their removal from the dead animal by dropping them into Dry Ice. The frozen glands (18.7 kilograms) were ground in a motor-driven meat grinder. The ground gland was added to a mixture of 37.4 liters of Krebs-Ringer solution, a nutrient medium, made alkaline to pH 7.4 with sodium bicarbonate, and 108 grams of nicotinamide (cofactor) and 35.3 grams of ll-desoxycorticosterone added and mixed thoroughly for about five minutes. The suspension was transferred to an incubation tank and maintained at 37 degrees centigrade for two hours, during which time a mixture of 95 percent oxygen and five percent carbon dioxide was bubbled through the suspension at a rate of about eight liters per minute.

The material from the incubation step was extracted three times with liters of acetone over a period of twenty-four hours, spent gland being separated and discarded, acetone removed from the extract by distillation under reduced pressure, and a 0.5 percent aliquot portion taken. Fresh acetone was added to the aliquot of the -11. resulting aqueous suspension to give a solvent containing fifty percent acetone, which was then extracted with 100 milliliters of Skellysolve B (hexane). The Skellysolve B solution was back- 12 showed the presence of 9.0 glycogen units, or 0.158 unit per gram of tissue.

One hundred milligrams of desoxycorticosterone showed no measurable deposition activity washed with IOU-milliliter and '70-milliliter porwhen assayed by the same procedure. tions of forty percent acetone, the forty percent Example 8 acetone extracts being added to the original fifty percent acetone solution, while the hexane layer fifty-seven grams of hog adreflal glands, from was discarded. The acetone was removed from the Same lot as a Were mcubatefi the the combined solutions under reduced pressure. 0 f manner as glven m Example 7 wlljh To the resulting aqueous Suspension was added milligrams of 11-desoxy-ll-hydroxycorticosterone-quarter of its volume of acetone and the one zl-afietate p acmg the ll-desoxycortiacetone Solution extracted with one Q mi11i costerone 1n the nutritive liquid. Isolation of the liter and. two IO-milliliter portions of Skellysolve actlve material ylelded more than 15 glycogen B. The Skellysolve B extracts were back-washed 15 umts on assaywith two lo-milliliter portions of twenty perone undred milligrams of ll-desoxy-l'I-hycent acetone, and the acetone extracts combined, droxywortlcosterpne acetate gave no measurable while the hexane layer was discarded. The comglycogen deposltlon When assayed by the Same bined acetone extracts were extracted six times Procedurewith 35-milliliter portions of ethylene dichloride, xample 9 which was removed under reducedpressure, and Adremu glands were frozen in Dry Ice immedithe residue dissolved in 35 milliliters of ethyl ately afte removal fr the animal body The acetate. The ethyl acetate solution was washed glands were then ground in a meat grinder and successively with two seven-milliliter portions of incubated as in Example '7 under conditions as tw percent sodium bmarbonate solutlon, p indicated in the table below. The steroid to be seven-milliliter portions of a one percent SOdlllm oxidized was added as crystalline material or as carbonate solution, two fou er portions of a concentrated acetone solution, as was most suit- 0.5 normal hydrochloric acid, two four-milliliter able for the amount added. After incubation, portions of two percent sodium bicarbonate soluthe glands were extracted and the extracts astion and four four-milliliter portions of distilled sayed for potency by the glycogen assay method. water. Each of the aqueous extracts was back- The quantities of reactants and th results obextracted with fifteen milliliters of ethyl acetate tained are as follows:

TABLE gcrcent Incuba- Rate Glycogen Glycogen x Amount f'c rti ii Substance and Amount (Jo-factors 0100 Temp-1 Units Units/gm 5 5%; if. Tissue Ringer Time, Flow, C. Assay, Tissue, to i Hours l./mm Total assay Med 001 pounds 3 Control, Gland.... 2 0.1 37 0.158 gm 2 0.1 39 an o. e e. 1 50 gm. 2 0.1 so 105 2. 13.8 7 50 gm 2 o 1 as ea 1. a 3.0 50 gm 2 0.1 39 110 2. 2 5. 2 50 3 0.1 31 115 2. 3 3. 0 50 gm 2 0.1 39 20 o. 4 0.7 51 gm 2 0.1 39 15 0. as 0.25 25 kgi" 2 1.0 31 3,650 1.4 as 1 g k 2 1.0 31 3,820 2.1 5.4 50 gm 2 0.1 31 50 1. 0 50. o 2.5 kg, 3 0. 5 31 1, 88'.) 0.10 35.0 50 gm 100 m1... Opd. S, 25 mg... 2 0.1 37 1.2 21.6

DOC-Desoxycorticosterone.

Opd. S-1l-Desoxy-l7-hydroxycorticosterone. ATP-Adcnosine Triphosphate. NA-Nicotinamide. l7-HP17-Hydroxyprogesteronc. ACTH-Adrcnocorticotropic hormone.

l Crystalline corticosterone isolated from these runs by chromatography on magnesium silicate column and crystallization from acetone. 2 Crystalline 17-hydroxycorticosterone (Compound F) isolated as in 3 0.158 already subtracted from glycogen assay per gm. value before conversion percentage calculated.

and then discarded. The ethyl acetate solutions were combined and the ethyl acetate removed under reduced pressure. The residue was dried, dissolved in fifteen milliliters of anhydrous acetone, and transferred to cottonseed oil for assay. Assay by the liver glycogen deposition test (Pabst et al. 100. cit.) showed the presence of more than 21000 glycogen units of activity in the final mixture or 1.1 unit per gram of tissue.

Example 712 (Control) Fifty-seven grams of hog adrenal glands from the same lot as above were incubated as above, with the exception that no ll-desoxycorticosterone was added to the nutritive liquid. The active It is to be understood that the invention is not to be limited to the exact details of operation or exact compounds shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is there fore to be limited only by the scope of the appended claims.

We claim:

1. Process for the bio-oxidative conversion of adrenal cortical steroids which are unoxygenated in the eleven position to ll-oxygenated adrenal cortical steroids which includes the step of aerobically incubating an adrenal cortical steroid which is unoxygenated in the eleven position of the steroid nucleus with previously frozen comminuted mammalian adrenal gland tissue, in a material, when isolated by the same procedure, 7. substantially aqueous medium and separating the 13 ll-oxygenated adrenal cortical steroid from the incubation reactionrmixture.

2. Process of claim 1, wherein the mammalian adrenal gland is hog adrenalgland.

3. Process of claim 1, wherein .the temperature of th incubation is maintained between about twenty and forty degrees centrigrade.

4. Process of claim 1, wherein the starting steroid is ll-desoxycorticosterone and the steroid product is corticosterone.

5. Process of claim 1, wherein the starting steroid is 11- desoxy-l'l-hydroxycorticosterone (Compound S) and the steroid product is 17-.hydroxycorticosterone (Compound .1?)

6. Process of claim 1, wherein the. starting steroid is l7-hydroxyprogesterone.

7. A process for the production of adrenal cortex hormones from mammalian adrenal glands, which includes: incubating for a period of at least forty hours and at a temperature between about ten degrees centigrade and that temperature at which adrenal glands are destroyed by heat, a water suspension of mammalian adrenal glands; and thereafter adding solvent for the adrenal cortex hormones and extracting the hormones.

8. A process for the preparation of adrenal cortex hormones from mammalian adrenal glands, which includes: incubating for a period of at least about forty hours and at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat, a suspension of said glands in an aqueous medium containing up to about twenty percent of a bacteriostatic agent and thereafter adding solvent for th hormones and extracting the adrenal cortex hormones.

9. A process as defined in claim 8, wherein the temperature is maintained between about twenty degrees and about forty degrees centigrade.

10. A process as defined in claim 8, wherein the aqueous medium is an aqueous-alcoholic medium containing up to about twenty percent alcohol.

11. A process for the preparation of adrenal cortex hormones from mammalian adrenal glands, which includes: incubating for a period of at least about forty hours at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat and under aerating conditions, a water suspension of mammalian glands, and thereafter adding solvent for the hormones and extracting the adrenal cortex hormones.

12. A process for the preparation of adrenal cortex hormones from mammalian adrenal glands, which includes: incubating for a period of at least about forty hours at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat and while aerating at a rate up to about liters per kilogram per minute, a water suspension of a mammalian adrenal gland; and, thereafter adding solvent for the hormones and extracting the adrenal cortex hormones.

13. A process for the preparation of adrenal cortex hormones from mammalian adrenal glands, which includes: incubating for a period of at least about forty hours at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat, while aerating at a rate up to about 15 liters per kilogram of gland 14 per minute, -a suspension of said glands in an aqueous medium containing'up to about 20 percent of a bacteriostatic agent and, thereafter adding solvent for the hormones and extracting the adrenal cortex hormones.

14. A process for the preparation of adrenal cortex hormones from hog adrenal glands, which includes: incubating for a period of at least forty hours at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat, hog adrenal glands in a water suspension; and thereafter adding solvent for the hormones and extracting the adrenal cortex hormones.

15. A process for the preparation of adrenal cortex hormones from hog adrenal glands, which includes: incubating for a period of at least forty hours at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat, a suspension of said glands in an aqueous medium containing up to about twenty percent of a bacteriostatic agent; and, thereafter adding solvent for the hormones and extracting the adrenal cortex hormones.

16. A process as defined in claim 15, wherein the temperature is maintained between about twenty degrees and about forty degrees centigrade.

1'7. A process as defined in claim 15, wherein the aqueous medium is an aqueous-alcoholic medium containing up to about twenty percent alcohol.

18. A process for the preparation of adrenal cortex hormones from hog adrenal glands, which includes: incubating for a period of at least about forty hours at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat, under aerating conditions, hog adrenal glands in a water suspension; and, thereafter adding solvent for the hormones and extracting the adrenal cortex hormones.

19. A process for the preparation of adrenal cortex hormones from hog adrenal glands, which includes: incubating for a period of at least forty hours at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat, while aerating at a rate up to about 15 liters per kilogram per minute, hog adrenal glands in a water suspension; and, thereafter adding solvent for the hormones and extracting the adrenal hormones.

20. A process for the preparation of adrenal cortex hormones from hog adrenal glands, which includes: incubating for a period of at least about forty hours, at a temperature between about ten degrees centigrade and that temperature at which the adrenal cortex hormones are destroyed by heat, while aerating at a rate up to about 15 liters per kilogram of gland per minute, a suspension of said glands in an aqueous medium containing up to about twenty percent of a bacteriostatic agent; and, thereafter adding solvent for the hormones and extracting adrenal cortex hormones.

21. In a process for the preparation of adrenal cortex hormones from mammalian adrenal glands, the step which comprises: incubating a suspension of said glands in a twenty percent aqueous-alcoholic medium at a temperature of from about twenty to about forty degrees centigrade, while aerating at a rate of from about 0.2 to about 1.5 liters per kilogram of gland per minute, fora period of from about forty to about 144 hours, and thereafter separating adrenal cortex hormones from the resulting mixture.

22. In a process for the preparation of adrenal cortex hormones from hog adrenal glands, the method which includes: incubating a suspension of said glands in a twenty percent aqueousalcoholic medium at a temperature of from about twenty to about forty degrees centigrade, While aerating at a rate of from about 0.2 to about 1.5 liters per kilogram of gland per minute for a period of from about 40 to about 144 hours, and thereafter separating adrenal cortex hormones from the resulting mixture.

WILLIAM J. HAINES. MARVIN H. KUIZENGA.

16 References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 829,220 Manns Aug. 21, 1906 1,000,214 Vanderkleed Aug. 8, 1911 1,003,646 Ohliger Sept. 19, 1911 1,515,976 Stern Nov. 18, 1924 2,074,492 Swingle Mar. 23, 1937 OTHER REFERENCES Kuizenga in J. Biol. Chem, vol. 147, March 1943, pages 561-565.

Hechter in J. Am. Chem. Soc., September 1949, vol. 71, pages 3261-3262.

Hayano in Proc. Soc. Exptl. Biol. & Med., v01. 72, December 1949, pages 700-701. 

1. PROCESS FOR THE BIO-OXIDATIVE CONVERSION OF ADRENAL CORTICAL STERIODS WHICH ARE UNOXYGENATED IN THE ELEVEN POSITION TO 11--OXYGENATED ADRENAL CORTICAL STEROIDS WHICH INDLUDES THE STEP OF AEROBICALLY INCUBATING AN ADRENAL CORTICAL STEROID WHICH IS UNOXYGENATED IN THE ELEVEN POSITION OF THE STEROID NUCLEUS WITH PREVIOUSLY FROZEN COMMINUTED MAMMALIAN ADRENAL GLAND TISSUE, IN A SUBSTANTIALLY AQUEOUS MEDIUM AND SEPARATING THE 11-OXYGENATED ADRENAL CORTICAL STEROID FROM THE INCUBATION REACTION MIXTURE. 